KBTBD7 Antibody, FITC conjugated

What is KBTBD7 and why is it a significant research target?

KBTBD7 (Kelch repeat and BTB domain containing 7) is a protein first cloned in 2010 that functions as both a transcriptional activator and a substrate adaptor during ubiquitination processes . Research has revealed KBTBD7's involvement in several critical biological processes, including:

• Excessive inflammation following myocardial infarction

• Brain development

• Neurofibromin stability

• Non-small cell lung cancer (NSCLC) progression

KBTBD7 has gained significant research interest due to its role in regulating PTEN through ubiquitin-dependent degradation, subsequently activating EGFR/PI3K/AKT signaling . This mechanism appears critical in cancer progression, particularly in NSCLC where KBTBD7 expression correlates with histological type, P-TNM stage, lymph node metastasis, and tumor size .

What are the specifications and storage requirements for KBTBD7 Antibody, FITC conjugated?

KBTBD7 Antibody, FITC conjugated is available as a rabbit polyclonal antibody with the following specifications:

For optimal storage:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze/thaw cycles

• When reconstituted in sterile pH 7.4 0.01M PBS, the antibody remains stable for at least two weeks at 2-4°C

What applications is KBTBD7 Antibody, FITC conjugated suitable for?

KBTBD7 Antibody, FITC conjugated has been validated for several research applications:

When using this antibody for the first time in any application, researchers should perform optimization experiments to determine the ideal concentration for their specific experimental conditions . This is particularly important as the optimal antibody dilution may vary depending on the cell type, fixation method, and detection system used.

How can FITC-conjugated antibodies be evaluated for thermal stability and antigen binding?

Recent research has demonstrated a novel approach for evaluating FITC-conjugated antibodies using differential scanning fluorimetry (DSF) without requiring additional extrinsic dyes . This methodology provides several advantages:

• The FITC label itself serves as the fluorescent reporter during thermal denaturation

• Allows direct assessment of antibody stability after FITC conjugation

• Enables quantitative measurement of potential conformational changes induced by conjugation

For quantitative assessment of antigen binding following FITC conjugation, isothermal titration calorimetry (ITC) has proven valuable . The ITC approach:

• Provides precise measurement of binding affinity changes

• Quantifies potential alterations in binding site accessibility

• Detects subtle changes in binding thermodynamics (enthalpy and entropy)

Research indicates that moderate FITC labeling (up to 5 mol FITC/mol antibody) typically results in only minor changes in antigen binding affinity and capacity .

What strategies should be employed when using KBTBD7 antibodies to study its role in ubiquitination pathways?

When investigating KBTBD7's role in ubiquitination pathways, particularly its interaction with substrates like PTEN, researchers should consider the following experimental approaches:

• Co-immunoprecipitation (Co-IP): This technique has successfully verified KBTBD7's direct interaction with PTEN at the protein level . When designing Co-IP experiments:

  • Include appropriate negative controls (IgG controls)

  • Validate antibody specificity prior to Co-IP

  • Consider both forward and reverse Co-IP approaches

• Ubiquitination assays: To evaluate KBTBD7's effect on substrate ubiquitination:

  • Pre-treat cells with proteasome inhibitors (e.g., MG132 for 12 hours) before cell collection

  • Immunoprecipitate the substrate protein (e.g., PTEN)

  • Perform western blotting with anti-ubiquitin antibodies

  • Consider using HA-tagged ubiquitin constructs for improved detection specificity

• RT-PCR analysis: To distinguish between transcriptional and post-translational regulation:

  • Measure mRNA levels of potential substrates (e.g., PTEN) following KBTBD7 knockdown

  • Absence of changes at the mRNA level with concurrent protein level changes suggests post-translational regulation

How can researchers effectively use KBTBD7 Antibody, FITC conjugated for localization studies in cancer research?

For researchers studying KBTBD7's role in cancer progression, particularly in NSCLC, FITC-conjugated antibodies offer advantages for localization studies. Consider these methodological approaches:

• Co-localization studies:

  • Use KBTBD7 Antibody, FITC conjugated alongside antibodies against potential interacting proteins (e.g., PTEN, EGFR) labeled with spectrally distinct fluorophores

  • Employ confocal microscopy with appropriate channel separation to minimize bleed-through

  • Quantify co-localization using established metrics (Pearson's correlation coefficient, Manders' overlap coefficient)

• Tissue microarray analysis:

  • KBTBD7 expression has been effectively evaluated in NSCLC using immunohistochemical staining of tissue microarrays

  • For fluorescent applications, use FITC-conjugated KBTBD7 antibody with appropriate nuclear counterstains

  • Consider parallel analysis of markers for proliferation, invasion, and EGFR/PI3K/AKT pathway activation

• Live cell imaging considerations:

  • When using FITC-conjugated antibodies for live cell applications, minimize exposure times to reduce phototoxicity

  • Consider photobleaching characteristics of FITC when designing time-lapse experiments

  • For quantitative analysis, include appropriate calibration standards

What are common issues with FITC-conjugated antibodies and how can they be addressed?

FITC-conjugated antibodies, including KBTBD7 Antibody, FITC conjugated, may present several technical challenges:

• Photobleaching:

  • FITC is more susceptible to photobleaching than many other fluorophores

  • Mitigate by using antifade mounting media containing radical scavengers

  • Minimize exposure to excitation light during sample preparation and imaging

  • Consider using reducing agents like n-propyl gallate or DABCO in mounting media

• pH sensitivity:

  • FITC fluorescence is optimal at alkaline pH and decreases significantly below pH 7.0

  • Ensure buffers maintain pH 7.2-8.0 during staining procedures

  • For fixed samples, verify fixation method doesn't create acidic microenvironments

• Background fluorescence:

  • Autofluorescence in the FITC channel can be problematic, particularly in tissues with high collagen content

  • Implement background subtraction during image analysis

  • Consider specialized autofluorescence quenching reagents when needed

  • Use appropriate blocking solutions containing 1-5% serum proteins and 0.1-0.3% Triton X-100 for intracellular targets

How does the degree of FITC labeling affect antibody performance in KBTBD7 research?

The degree of FITC labeling (DOL) can significantly impact antibody performance. Research on FITC-labeled antibodies has revealed :

• Effects on antigen binding:

  • Low to moderate labeling (1-5 mol FITC/mol antibody) generally preserves antigen binding properties

  • Higher labeling can decrease antigen binding affinity and capacity

  • At excessive labeling levels, conformational changes may occur that disrupt antibody function

• Distribution of FITC labels:

  • FITC preferentially labels the Fab portion of antibodies at lower DOL

  • As DOL increases, labeling becomes more distributed across the entire antibody molecule

  • Lysine residues near antigen binding sites may affect binding when labeled, even if not directly in the binding pocket

• Optimization strategies:

  • Titrate antibody concentration for each application to determine optimal working dilution

  • Consider using F(ab')2 fragments for applications where Fc-mediated effects are problematic

  • For quantitative applications, characterize each lot for DOL and standardize protocols accordingly

What alternative approaches can complement or enhance KBTBD7 Antibody, FITC conjugated studies?

To enhance research with KBTBD7 Antibody, FITC conjugated, consider these complementary approaches:

• Genetic manipulation techniques:

  • siRNA or shRNA knockdown of KBTBD7 has demonstrated effective suppression of KBTBD7 expression in NSCLC cell lines

  • CRISPR-Cas9 genome editing can provide more stable and complete KBTBD7 knockout models

  • Overexpression systems using tagged KBTBD7 constructs can help identify novel interacting partners

• Functional assays to assess biological significance:

  • Proliferation assays: KBTBD7 knockdown inhibits NSCLC cell proliferation

  • Invasion assays: KBTBD7 suppression reduces invasive capacity of cancer cells

  • Signaling pathway analysis: Monitor EGFR/PI3K/AKT pathway components (phosphorylation status) following KBTBD7 manipulation

• Molecular interaction studies:

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Proximity ligation assay (PLA) for in situ detection of protein-protein interactions

  • Mass spectrometry-based approaches to identify novel KBTBD7-interacting proteins or substrates

How can KBTBD7 Antibody, FITC conjugated be used to investigate its role in the CUL3 ubiquitin ligase complex?

KBTBD7 functions as a substrate adaptor for CUL3-based E3 ubiquitin ligase complexes . To investigate this role:

• Vesicular trafficking analysis:

  • Research has revealed that CUL3-KBTBD6/KBTBD7 is recruited to GABARAP-containing vesicles to regulate membrane-associated TIAM1

  • Use FITC-conjugated KBTBD7 antibody alongside markers for vesicular compartments (early endosomes, late endosomes, lysosomes)

  • Implement live-cell imaging to track dynamic recruitment to vesicular structures

• Substrate identification approaches:

  • Combine KBTBD7 immunoprecipitation with mass spectrometry to identify novel substrates

  • Verify interactions using reciprocal Co-IP and in vitro binding assays

  • For confirmed interactions, design ubiquitination assays as described in section 2.2

• Structural studies:

  • The BTB domain mediates interaction with CUL3, while the Kelch repeat domain typically recognizes substrates

  • Use domain-specific antibodies or constructs to dissect the functional importance of each region

  • Consider implementing proximity-based labeling methods (BioID, APEX) to map the local interactome

What considerations are important when using KBTBD7 Antibody, FITC conjugated in multiplex imaging experiments?

For multiplex imaging involving KBTBD7 Antibody, FITC conjugated:

• Spectral compatibility:

  • FITC has excitation/emission maxima at approximately 495/519 nm

  • Compatible fluorophores for multiplex imaging include:

    • TRITC/Cy3 (excitation/emission: ~550/570 nm)

    • Cy5 (excitation/emission: ~650/670 nm)

    • DAPI (excitation/emission: ~358/461 nm) for nuclear counterstaining

• Signal optimization:

  • When designing multiplex panels, consider relative abundance of targets

  • Assign brightest fluorophores to least abundant targets

  • FITC may not be ideal for low-abundance targets due to photobleaching characteristics

  • Implement appropriate controls for spectral unmixing

• Technical considerations:

  • Sequential imaging may be necessary to minimize bleed-through

  • Consider cross-reactivity when using multiple primary antibodies from the same host species

  • For tissue sections, implement autofluorescence reduction strategies specific to FITC channel

How can researchers integrate KBTBD7 antibody studies with pathway analysis to elucidate its role in cancer progression?

KBTBD7 has been implicated in cancer progression through its effects on the EGFR/PI3K/AKT pathway . For comprehensive pathway analysis:

• Multi-parameter phosphoprotein analysis:

  • Following KBTBD7 manipulation, assess phosphorylation status of:

    • EGFR (pY1068, pY1173)

    • AKT (pS473, pT308)

    • Downstream effectors (pS6, p4EBP1)

  • Correlate findings with changes in cellular phenotypes (proliferation, invasion)

• Transcriptomic analysis:

  • RNA-seq following KBTBD7 knockdown can identify gene expression changes beyond direct protein interactions

  • Pathway enrichment analysis may reveal broader regulatory networks

  • Validate key findings at the protein level using immunoblotting or immunofluorescence

• Clinical correlation studies:

  • KBTBD7 expression has been positively correlated with histological type, P-TNM stage, lymph node metastasis, and tumor size in NSCLC

  • Design tissue microarray studies using FITC-conjugated KBTBD7 antibody alongside markers for:

    • Cell cycle regulation (CCNE1, CDK4, P27)

    • Epithelial-mesenchymal transition (ZEB-1, E-cadherin)

    • Cell invasion (Claudin-1, ROCK1, MMP-9)

What novel therapeutic approaches might target KBTBD7 in cancer treatment?

Based on KBTBD7's role in NSCLC progression, several therapeutic approaches warrant investigation:

• Small molecule inhibitors:

  • Target the BTB domain to disrupt CUL3 interaction

  • Target the Kelch repeat domain to prevent substrate recognition

  • Rational drug design based on structural insights could yield specific inhibitors

• Degrader technologies:

  • Proteolysis-targeting chimeras (PROTACs) could redirect KBTBD7 for degradation

  • Molecular glue degraders might enhance KBTBD7 recruitment to other E3 ligases

• Combination therapies:

  • Since KBTBD7 activates EGFR/PI3K/AKT signaling through PTEN degradation , combination with:

    • EGFR inhibitors (erlotinib, gefitinib)

    • PI3K inhibitors (alpelisib, copanlisib)

    • AKT inhibitors (capivasertib, ipatasertib)

  • May show synergistic effects in KBTBD7-overexpressing tumors

Research has indicated that KBTBD7 "may be a potential therapeutic target for the treatment of NSCLC" , making these approaches particularly relevant for translational cancer research.

How might KBTBD7 Antibody, FITC conjugated contribute to understanding the role of KBTBD7 in inflammation?

KBTBD7 has been implicated in promoting inflammatory responses, particularly after myocardial infarction . FITC-conjugated antibodies could advance this research through:

• Inflammatory cell analysis:

  • Track KBTBD7 expression in macrophages and other inflammatory cells

  • Correlate with production of inflammatory cytokines (IL-1β, IL-6, TNF-α)

  • Use flow cytometry with FITC-conjugated KBTBD7 antibody to quantify expression across immune cell populations

• Signaling pathway investigation:

  • KBTBD7 knockdown significantly inhibits p38 and NF-κB signaling pathways

  • Multiplex imaging with FITC-conjugated KBTBD7 antibody and phospho-specific antibodies against p38 MAPK and NF-κB pathway components

  • Correlate KBTBD7 expression with nuclear translocation of NF-κB

• Therapeutic modulation studies:

  • Crocin has been shown to inhibit KBTBD7, preventing excessive inflammation and cardiac dysfunction following myocardial infarction

  • Investigate other potential KBTBD7 inhibitors using cell-based inflammatory models

  • Assess therapeutic potential through quantification of inflammatory markers following KBTBD7 modulation